Substrate having an electron donating surface with metal particles comprising palladium on said surface

ABSTRACT

There is disclosed a substrate with an electron donating surface, characterized in having metal particles on said surface, said metal particles comprising palladium and at least one metal selected from the group consisting of gold, ruthenium, rhodium, osmium, iridium, and platinum, wherein the amount of said metal particles is from about 0.001 to about 8 μg/cm 2 . Examples of coated objects include contact lenses, pacemakers, pacemaker electrodes, stents, dental implants, rupture nets, rupture mesh, blood centrifuge equipment, surgical instruments, gloves, blood bags, artificial heart valves, central venous catheters, peripheral venous catheters, vascular ports, haemodialysis equipment, peritoneal dialysis equipment, plasmapheresis devices, inhalation drug delivery devices, vascular grafts, arterial grafts, cardiac assist devices, wound dressings, intermittent catheters, ECG electrodes, peripheral stents, bone replacing implants, orthopaedic implants, orthopaedic devices, tissue replacing implants, intraocular lenses, sutures, needles, drug delivery devices, endotracheal tubes, shunts, drains, suction devices, hearing aid devices, urethral medical devices, and artificial blood vessels.

FIELD OF THE INVENTION

The present invention relates to a new substrate with nano particles,which makes it possible to modify surface properties relating tobiocompatibility as well as antimicrobial properties in a repeatable andcontrolled manner. Examples of surface properties, which can bemodified, include but are not limited to hydrophobicity, proteinadsorption, adhesion of bacteria, tissue ingrowth, complementactivation, inflammatory response, thrombogenicity, frictioncoefficient, and surface hardness. Examples of uses of the substrateinclude but are not limited to preventing transmission of bacteria andin particular nosocomial infections. The present invention furtherrelates to objects comprising said new substrate. The present inventionfurther relates to the use of said substrate. Finally the presentinvention further relates to a method for the manufacture of such asubstrate.

BACKGROUND

It has always been desirable to modify surface characteristics toachieve useful properties. In particular it is desired to be able tomodify surface properties that are important in connection withantimicrobial and biocompatible objects. Examples of known surfacemodifications for different purposes are outlined below.

U.S. Pat. No. 6,224,983 discloses an article with an adhesive,antimicrobial and biocompatible coating comprising a layer of silverstabilised by exposure to one or more salts of one or more metalsselected from the group consisting of platinum, palladium, rhodium,iridium, ruthenium and osmium. The thickness of the silver layer is inthe range 2-2000 Å (Angström, Angstrom, 10⁻¹⁰ m) and further disclosedranges are 2-350 Å and 2-50 Å. There are also examples of a thickness ofthe silver layer of 50 Å, 350 Å, 500 Å, and 1200 Å. The substrate may belatex, polystyrene, polyester, polyvinylchloride, polyurethane, ABSpolymers, polycarbonate, polyamide, polytetrafluoroethylene, polyimideor synthetic rubber.

U.S. Pat. No. 5,965,204 discloses a method for preparing an article madeof a nonconducting substrate having a coating comprising a silver layer,which has been deposited after activating the surface with stannousions. There is also disclosed a coating further comprising a platinumgroup metal or gold. The thickness of the silver layer is in the range2-2000 Å and further disclosed ranges are 2-350 Å and 2-50 Å. There arealso examples of a thickness of the silver layer of 50 Å, 350 Å, 500 Å,and 1200 Å.

U.S. Pat. No. 5,747,178 discloses an article made by depositing a silverlayer. The layer is said to be adhesive, antimicrobial andbiocompatible. The silver layer may be stabilized by exposure to a saltsolution of one or more platinum group metals or gold. The thickness ofthe silver layer is in the range 2-2000 Å and further disclosed rangesare 2-350 Å and 2-50 Å. There are also examples of a thickness of thesilver layer of 50 Å, 350 Å, 500 Å, and 1200 Å. The article may be madeof latex, polystyrene, polyester, polyvinylchloride, polyurethane, ABSpolymers, polycarbonate, polyamide, polytetrafluoroethylene, polyimideor synthetic rubber.

U.S. Pat. No. 5,395,651 discloses a method of preparing an antimicrobialdevice comprising a nonconducting material with a silver coating. Thecoating also comprises a platinum group metal and/or gold. The methodcomprises the steps: 1 activating the surface to be coated; 2 depositingsilver on the surface; 3 treating the surface with a salt of a platinumgroup metal and/or gold, which is to be carried out for only sufficienttime to result in a thin coating; 4 rinsing with water. The treatment ofstep 3 can utilise a salt of platinum or palladium in combination withgold. Nothing is said about the thickness of the coating of the platinumgroup metal and/or gold. The coating is only described as a thincoating. Nothing is said about metal particles on the silver coating.The thickness of the silver layer is in the range 2-2000 Å and furtherdisclosed ranges are 2-350 Å and 2-50 Å. There are also examples of athickness of the silver layer of 50 Å, 350 Å, 500 Å, and 1200 Å.

U.S. Pat. No. 5,320,908 discloses an adhesive, antimicrobial, andbiocompatible coating consisting essentially of a layer of silveroverlaid by one of more platinum group metals or gold. The coating maybe transparent to the human eye. The thickness of the silver layer is inthe range 2-2000 Å and further disclosed ranges are 2-350 Å and 2-50 Å.There are also examples of a thickness of the silver layer of 50 Å, 350Å, 500 Å, and 1200 Å. The article may be made of latex, polystyrene,polyester, polyvinylchloride, polyurethane, ABS polymers, polycarbonate,polyamide, polytetrafluoroethylene, polyimide or synthetic rubber.

U.S. Pat. No. 5,695,857 discloses antimicrobial surfaces with severallayers of one first metal and a second nobler metal. The antimicrobialactive metal may for instance be platinum, gold, silver, zinc, tin,antimony and bismuth. The nobler metal may for instance be selected fromthe group consisting of platinum, osmium, iridium, palladium, gold,silver and carbon. The surface is to be used with biological fluids andeach of the layers not in contact with the substrate are discontinuousso that the layer below is exposed. One example of a surface is silvercoated with gold or platinum. Other examples are copper in combinationwith silver, copper in combination with a copper silver alloy, copper incombination with gold or a silver copper alloy in combination with gold.

CH 654 738 A5 discloses surgical implants made of stainless steel, whichis coated with a first layer of copper and a second layer of silver,gold, rhodium or palladium. Silver is described to have a bactericidicaction. CH 654 738 A5 explicitly discloses a surface where stainlesssteel is coated with 10 μm copper and 5 μm (50 000 Å) palladium. Allsurfaces disclosed in CH 654 738 A5 have a layer of 10 μm copper (100000 Å) and either 10 μm silver or 5 μm gold or 5 μm palladium.

WO 2005/073289 discloses fibres made of a polymer composite comprisingmetal nanoparticles. It is stated that many metals have antimicrobialeffects. Antimicrobial fibres are mentioned. One example is ahydrophilic fibre used in antimicrobial would dressings. Fibres withantimicrobial properties can comprise Ag, Au, Pt, Pd, Ir, Sn, Cu, Sb, Bior Zn or any combination thereof.

SHORT SUMMARY OF THE PRESENT INVENTION

A problem in the state of the art regarding surfaces is how to provide asurface which for example is antimicrobial and biocompatible, wherein itin a repeatable way is possible to modify the hydrophobicity, proteinadsorption, adhesion of bacteria, tissue ingrowth, complementactivation, inflammatory response, thrombogenicity, frictioncoefficient, and surface hardness.

The present inventors have discovered that the above-mentioned problemin the state of the art is solved by a substrate having an electrondonating surface, characterized in that there are metal particles onsaid surface, said metal particles comprise palladium and at least onemetal selected from the group consisting of gold, ruthenium, rhodium,osmium, iridium, and platinum and wherein the amount of said metalparticles is from about 0.001 to about 8 μg/cm². Further embodiments ofthe present invention are defined in the appended dependent claims,which hereby are incorporated by reference.

DESCRIPTION Definitions

Before the invention is disclosed and described in detail, it is to beunderstood that this invention is not limited to particularconfigurations, process steps and materials disclosed herein as suchconfigurations, process steps and materials may vary somewhat. It isalso to be understood that the terminology employed herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention islimited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise.

The following terms are used throughout the description and the claims.

“Adhesion of bacteria” as used herein is the phenomenon where bacteriaadhere to a surface.

“Antimicrobial” as used herein is the property of suppressing oreliminating microbial growth.

“Biocompatible” as used herein is the ability of a material to performwith an appropriate host response in a specific application.

“Biofilm” as used herein is a thin layer in which microorganisms areembedded. Biofilms occur when microorganisms colonise a surface.

“Complement activation” as used herein is a complex system of factors inblood plasma that may be activated by a chain reaction from component C1to C9, which give rise to a number of biological effects. Complementactivation occurs in two ways a) the classical C1 to C9, or b) thealternative by direct activation of C3.

“Contact angle”. For a given droplet on a solid surface the contactangle is a measurement of the angle formed between the surface of asolid and the line tangent to the droplet radius from the point ofcontact with the solid.

“Electron donating material” as used herein is a material, which inconnection with another more noble material has the ability to transferelectrons to the more noble material. An example is a less noble metaltogether with a more noble metal.

“Electron donating surface” as used herein is a surface layer comprisingan electron donating material.

“Hydrophobicity” of a surface as used herein describes the interactionsbetween the surface and water. Hydrophobic surfaces have little or notendency to adsorb water and water tends to “bead” on their surfaces.The term hydrophobicity of a surface is also closely linked with itssurface energy. Whereas surface energy describes interactions of thesurface with all molecules, the hydrophobicity describes theinteractions of the surface with water.

“Hysteresis of contact angle” as used herein is the difference betweenthe advancing and receding contact angle values. The advancing contactangle of a drop of water on a surface is the contact angle when theboundary between water and air is moving over and wetting the surface,while the receding angle is the contact angle when boundary betweenwater and air is withdrawn over a pre-wetted surface.

“Inflammatory response” occurs when tissues are injured by viruses,bacteria, trauma, chemicals, heat, cold or any other harmful stimulus.Chemicals including bradykinin, histamine, serotonin and others arereleased by specialised cells. These chemicals attract tissuemacrophages and white blood cells to localise in an area to engulf anddestroy foreign substances.

“Modify” either means reducing or enhancing a property.

“Noble” is used herein in a relative sense. It is used to relatematerials including metals to each other depending on how they interactwith each other. When two metals are submerged in an electrolyte, whileelectrically connected, the term “less noble” metal is used to denotethe metal which experiences galvanic corrosion. The term “more noble” isused to denote the other metal. Electrons will be transferred from the“less noble” metal to the more noble metal.

“Nosocomial infection” as used herein describes an infectious diseasespreading in a hospital environment.

“Protein adsorption” as used herein is the phenomenon where proteinsadhere to a surface due to overall attractive forces between theproteins and the surface.

“Substrate” as used herein is the base, which is treated according tothe present invention.

“Tissue ingrowth” is the process where cells start to grow on a surface,forming new tissue.

“Thrombogenicity” as used herein is the ability of a substrate to induceclotting of blood.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

According to the present invention a substrate is treated to give itdesired properties. The substrate can be made of a wide range ofmaterials. In one embodiment the substrate is made of a material, whichhas an electron-donating surface. In an alternative embodiment it ismade of a material, which does not have an electron-donating surface. Inthe case of an electron-donating surface the metal particles can beapplied directly on to the electron-donating surface. In the case wherethe surface it not electron donating, a layer of an electron donatingmaterial has to be applied to create an electron donating surface.

The present invention comprises a substrate having an electron donatingsurface, characterized in that there are metal particles on saidsurface, said metal particles comprise palladium and at least one metalselected from the group consisting of gold, ruthenium, rhodium, osmium,iridium, and platinum and wherein the amount of said metal particles isfrom about 0.001 to about 8 μg/cm². A preferred amount of said metalparticles is from about 0.01 to about 4 μg/cm². A particularly preferredamount of said metal particles is from about 0.01 to about 1 μg/cm².Examples of ranges within from about 0.001 to about 8 μg/cm² include0.001-6, 0.001-4, 0.001-2, 0.001-1, 0.001-0.5, 0.001-0.25, 0.001-0.15,0.15-8, 0.25-8, 0.5-8, 1-8, 2-8, 4-8, 6-8, 0.15-0.25, 0.25-0.5, 0.5-1,1-2, 2-4, 4-6 1-3, and 3-6 μg/cm².

Either the substrate itself is electron donating or there is applied alayer of an electron donating material on the substrate. In the casewhere the electron donating material is applied on the substrate it isapplied in an amount of from about 0.05 to about 12 μg/cm². The amountof the substrate can also be within other ranges as long as the amountis from about 0.05 to about 12 μg/cm². Examples of such other rangesinclude 0.05-10, 0.05-8, 0.05-6, 0.05-4, 0.05-2, 0.05-1, 0.05-0.5,0.05-0.25, 0.05-0.15, 0.15-12, 0.25-12, 0.5-12, 1-12, 2-12, 4-12, 6-12,8-12, 10-12, 0.15-0.25, 0.25-0.5, 0.5-1, 1-2, 2-4, 4-6, 6-8, 8-10, 1-5,and 5-10 μg/cm².

An electron donating material does not necessarily have anelectron-donating surface. An example is aluminium, which in air gets anoxide layer, which is not an electron-donating surface.

The electron donating material is any material with the ability to forman electron-donating surface, such as a conducting polymer or a metal.In the case of a metal it must be less noble than any of the metals inthe group consisting of palladium, gold, ruthenium, rhodium, osmium,iridium, and platinum.

A preferred metal for use as an electron-donating surface is a metalselected from the group consisting of silver, copper and zinc.

In one embodiment of the present invention the substrate is a polymericsubstrate.

In one embodiment the substrate is selected from the group consisting oflatex, vinyl, polymers comprising vinyl groups, polyurethane urea,silicone, polyvinylchloride, polypropylene, styrene, polyurethane,polyester, copolymerisates of ethylene vinyl acetate, polystyrene,polycarbonate, polyethylene, polyacrylate, polymethacrylate,acrylonitrile butadiene styrene, polyamide, and polyimide, or mixturesthereof.

In another embodiment of the present invention the substrate is selectedfrom the group consisting of a natural polymer, a degradable polymer, anedible polymer, a biodegradable polymer, an environmental friendlypolymer, and a medical grade polymer.

In another embodiment of the present invention the substrate is a metal.

A preferred metal for the substrate is selected from the groupconsisting of stainless steel, medical grade steel, titanium, medicalgrade titanium, cobalt, chromium and aluminium or mixtures thereof.

In another embodiment of the present invention the substrate is selectedfrom the group consisting of glass, minerals, zeolites, stone andceramics.

In another embodiment of the present invention the substrate is selectedfrom the group consisting of paper, wood, woven fibres, fibres,cellulose fibres, leather, carbon, carbon fibres, graphite,polytetrafluoroethylene, and polyparaphenyleneterephthalamide.

In another embodiment of the present invention the substrate has theshape of a particle.

In one embodiment of the present invention there is provided an objectcomprising a substrate according to the present invention. Examples ofobjects comprising a substrate according to the present inventioninclude medical devices, medical instruments, disposable articles,medical disposable articles. Further examples of objects comprising asubstrate coated according to the present invention include contactlenses, pacemakers, pacemaker electrodes, stents (bare metal and drugeluting), dental implants, rupture nets, rupture mesh, blood centrifugeequipment (in contact with blood), surgical instruments, gloves, bloodbags, artificial heart valves, central venous catheters, peripheralvenous catheters, vascular ports, haemodialysis equipment, peritonealdialysis equipment, plasmapheresis devices, inhalation drug deliverydevices, vascular grafts, arterial grafts, cardiac assist devices, wounddressings, intermittent catheters, ECG electrodes, peripheral stents,bone replacing implants, orthopaedic implants, orthopaedic devices(screws, pins, staples, suture anchors etc.), tissue replacing implants,intraocular lenses, sutures, needles, drug delivery devices,endotracheal tubes, shunts, drains, suction devices, hearing aiddevices, urethral medical devices, and artificial blood vessels.

The particles must always comprise palladium. In addition to palladiumthere is at least one other metal. A ratio of palladium to other metalsin the metal particles of from about 0.01:99.99 to about 99.99:0.01 canbe used in the present invention. A ratio from about 0.5:99.5 to about99.8:0.2 is preferred. Particularly preferred ratios are from about 2:98to about 95:5. Very particularly preferred ratios are 5:95 to 95:5. Inanother embodiment the ratios are from about 10:90 to about 90:10. Aperson skilled in the art realises that the ration also can be in otherintervals. Examples of other ranges for the ratio include: 0.01:99.99 to0.05:99.95, 0.05:99.95 to 0.1:99.9, 0.1:99.9 to 0.5:99.5, 0.5:99.5 to1:99, 1:99 to 2:98, 2:98 to 4:96, 4:96 to 6:94, 6:94 to 8:92, 8:92 to10:90, 10:90 to 20:80, 20:80 to 30:70, 30:70 to 40:60, 40:60 to 50:50,50:50 to 60:40, 60:40 to 70:30, 70:30 to 80:20, 80:20 to 90:10, 90:10 to92:8, 92:8 to 94:6, 94:6 to 96:4, 96:4 to 98:2, 98:2 to 99:1, 99:1 to99.5:0.5, 99.5:0.5 to 99.9:0.1 to 99.95:0.05, 99.95:0.05 to 99.99:0.01

In one embodiment of the present invention said metal particles, inaddition to palladium, comprise gold.

The present inventors have discovered that advantageous properties areachieved when said metal particles have an average size of from about 10to about 10000 Å.

In one embodiment the average sizes for said metal particles are fromabout 100 to about 600 Å.

A person skilled in the art realises that the particle size can be indifferent intervals within from about 10 to about 10000 Å. Examples ofsuch intervals include 10-8000 Å, 10-6000 Å, 10-4000 Å, 10-2000 Å,10-1000 Å, 10-100 Å, 100-10000 Å, 1000-10000 Å, 2000-10000 Å, 4000-10000Å, 6000-10000 Å, 8000-10000 Å, 100-1000 Å, 1000-2000 Å, 2000-4000 Å,4000-6000 Å, 6000-8000 Å, 1000-5000 Å, and 5000-8000 Å.

In another aspect of the present invention there is provided an objectcomprising any of the substrates described herein.

There is also provided a medical device comprising any of the substratesdescribed herein.

A disposable article comprising any of the substrates described hereinis also provided.

The present invention also provides a dental article, as well as dentalequipment, dental implants, and dental devices, comprising any of thesubstrates described herein.

The applied amount of the metal particles is expressed in μg/cm² and itmust be realised that the metal particles do not form a covering layer,but instead are uniformly distributed particles or clusters on saidelectron donating surface.

An applied layer of an electron donating material is preferably appliedso that it is uniform, essentially without agglomerates or clusters onthe surface. If the electron donating surface layer is homogenous anduniform the applied amount in μg/cm² may be converted to a thickness inÅ. An applied amount of 0.05-4 μg/cm² corresponds to about 4.8-380 Å,0.5-8 μg/cm² corresponds to about 48-760 Å, and 0.8-12 μg/cm²corresponds to about 76-1140 Å.

In one embodiment of the present invention the electron-donating surfaceis a layer of commercially available essentially pure silver, which doesnot exclude the possibility of small amounts of impurities.

If the substrate does not have an electron donating surface and thus adeposition of an electron donating surface layer is necessary, thedeposition is performed using a method selected from the groupconsisting of chemical vapour deposition, sputtering, and deposition ofmetal from a solution comprising a metal salt. A uniform layeressentially without clusters or agglomerates is the result of thedeposition. Preferably the deposition is carried out so that the firstlayer has good adhesion to the substrate.

Now there is described one embodiment of the present invention forpreparation of the coated substrate. For substrates which do not have anelectron donating surface the method includes some or all of thefollowing steps:

1. pre-treatment2. rinsing3. activation4. deposition of an electron donating surface5. rinsing6. deposition of metal particles7. rinsing8. drying

For objects with an electron-donating surface the method comprises thesteps

1. rinsing2. deposition of metal particles3. rinsing4. drying

In the following, one embodiment of steps 1 to 9 for substrates which donot have an electron-donating surface is described more in detail.

The pre-treatment can be made in an aqueous solution of a stannous saltcontaining 0.0005 to 30 g/l of stannous ions. The pH is 1 to 4 andadjusted by hydrochloric and/or sulphuric acid. The treatment time is2-60 minutes at room temperature. After the pre-treatment the surface isrinsed in demineralised water, but not dried.

The activated and rinsed substrate is transferred to the depositionsolution. The deposition solution has a pH of not less than 8. Itincludes a metal salt selected from the group consisting of a silversalt, a zinc salt, and a copper salt. In one embodiment of the presentinvention the salt is silver nitrate (AgNO₃). The metal salt is used inan effective amount of no more than about 0.10 grams per litre,preferably about 0.015 grams per litre. If the metal content is aboveabout 0.10 grams per litre, the elemental metal may form nonuniformly,in the solution or on the container walls. If the metal content is belowan effective amount, there is insufficient metal to form a film in thedesired time.

A second component of the deposition solution is a reduction agent thatreduces the metal-containing salt to elemental metal. The reductionagent must be present in an amount sufficient to accomplish the chemicalreduction. Acceptable reduction agents include formaldehyde, hydrazinesulphate, hydrazine hydroxide, and hypo phosphoric acid. In oneembodiment of the present invention it is present in an amount of about0.001 millilitres per litre of solution. Too large a concentration ofthe reduction agent causes deposition of metal throughout the solutionand on the container walls, while too small a concentration may resultin an insufficient formation of metal on the substrate. A person skilledin the art can in the light of this description by routineexperimentation determine the desired amount of reduction agent.

Another component of the deposition solution is a deposition controlagent that is present in an amount sufficient to slow the depositionreaction to prevent the reduced metal from precipitating directly fromsolution as a fine metallic powder, or precipitating onto the walls ofthe container. Operable deposition control agents include invertedsugar, also known as invertose, succinic acid, sodium citrate, sodiumacetate, sodium hydroxide, potassium hydroxide, sodium tartrate,potassium tartrate, and ammonia. The deposition control agent ispreferably present in an amount of about 0.05 grams per litre ofsolution. If too little is present, there may occur precipitation ofmetal clusters instead of a uniform metallic surface. If too much ispresent, the metal-containing salt may become too stable for the desiredprecipitation onto the substrate of interest.

The concentrations of the reduction agent and the deposition controlagent are adjusted as necessary to achieve the desired results,depending upon the substrate material, the thickness of the filmdesired, the conditions of deposition, and the concentration of metal inthe solution. For example, for thin films the metal salt concentrationwill be relatively low, as will the concentrations of the reductionagent and the deposition control agent. A person skilled in the art canin the light of this description by routine experimentation determinethe desired amount of deposition control agent.

In preparing the deposition solution, each of the components of thesolution are preferably individually dissolved in demineralised water.The various pre-solutions are then mixed, and diluted where necessary,in the correct amounts to achieve the concentrations mentioned above.

The combination of a metal salt and reduction agent permits the metal tobe reduced from the salt in a suitable state to be deposited upon thesurface of the substrate. This method is particularly beneficial toachieve good adhesion of the completed metal film to the substratesurface. Good adhesion is important in nearly all uses.

The substrate surface is exposed to the deposition solution by anyappropriate procedure. Dipping into the solution is normally preferred,but the solution may be applied by any convenient technique such asspraying or brushing. The metal film deposits uniformly from thesolution at a rate that may be controlled by the concentration of themetal salt. If a thin film is required, the temperature of deposition ismaintained sufficiently low so that deposition is controllably slow.

Other methods of applying a metal layer that acts as anelectron-donating surface can also be applied in the present invention.Other ways of achieving an electron-donating surface are chemical vapourdeposition and sputtering.

After the above-described metal deposition the substrate has anelectron-donating surface consisting of a metal. This metal depositionis only necessary if the substrate does not have an electron-donatingsurface from the start. If the substrate already possesses anelectron-donating surface, metal particles can be deposited on thesurface without the extra addition of a metal layer. In the latter casethe substrate is cleaned thoroughly before application of the particles.

The next step in the manufacturing method is deposition of metalparticles.

In one embodiment colloidal suspensions of metals are used to obtainparticles comprising palladium and at least another metal on thesurface. The metal particles are deposited from a suspension of thedesired particles. The composition of the metal particles in thesuspension is adjusted according to the preferred value. The substratewith the electron-donating surface is dipped in the suspension of metalparticles for a period of time from about a few seconds to about a fewminutes or longer.

The suspension of metal particles can be manufactured in several ways.In one embodiment the suspension of metal particles is made from anaqueous solution of a metal salt which is reduced under conditions suchthat metal particles of a desired size are formed. Mixing a suitableamount of metal salt, reducing agent and stabilising agent achievesthis. The same reducing agents and stabilising agents as described abovecan be used when making the particle suspension. A person skilled in theart can in the light of this description by routine experimentationdetermine the desired amount of reducing agent and stabilising agent toget the desired particle size. In an alternative embodiment acommercially available colloidal suspension of metal particles is used.Metal particles of the desired composition are used to make thesuspension.

In one embodiment the suspension of metal particles is made by dilutingwith demineralised water a commercially available concentrated colloidalsolution of metal particles comprising palladium and at least one metalselected from the group consisting of gold, ruthenium, rhodium, osmium,iridium, and platinum. The substrate is treated with the suspension fora period of time from about a few seconds to about a few minutes orlonger. After the treatment the substrate is rinsed in a solvent orwater such as demineralised water and left to dry in room temperature.

In one particular non-limiting embodiment the commercially availablemetal particles consist of 75% palladium and 25% gold.

Thus according to the present invention, a substrate with a particulardesired surface can be obtained. For example, one can prepare asubstrate having a silver electron donating surface with particlesconsisting of 75% palladium and 25% gold, or a copper electron donatingsurface with particles consisting of 85% palladium and 15% ruthenium.

One of the advantages offered by the flexible yet controlled andrepeatable method for producing such substrates is that a wide varietyof substrates can be produced. As described further herein, certainsubstrates have improved properties over existing substrates. Forexample a particular substrate according to the present invention canproduce surprising and advantageous modifications of the hydrophobicityof a substrate to which is it applied. Other properties that can bemodified in this way by substrates according to claim 1 include proteinadsorption, adhesion of bacteria, tissue ingrowth, complementactivation, inflammatory response, thrombogenicity, frictioncoefficient, and surface hardness.

That is, it is possible to adjust the particle size, the composition ofthe particles and the amount of particles to modify the surfaceproperties of objects to which the substrate is applied.

The present inventors have discovered that it is possible to achievethis by using a substrate according to claim 1. In particular it ispossible to adjust the particle size, the composition of particles, andthe amount of particles to modify the surface properties.

Substrates according to the present invention can be used for manypurposes. They are suitable for use in any application where it isdesired to modify hydrophobicity, protein adsorption, adhesion ofbacteria, tissue ingrowth, complement activation, inflammatory response,thrombogenicity, friction coefficient, and surface hardness of asubstrate.

Properties of the substrate can be both reduced or increased. Thusobjects are provided which display at least one area which enhances afeature, and at least one area which reduces a feature. An example is anobject with an area that reduces protein adsorption and an area thatenhances protein adsorption. Another example is an object with an areathat reduces tissue ingrowth and an area that enhances tissue ingrowth.

A substrate according to the present invention also comprises asubstrate having an electron donating surface, with metal particles onsaid surface, said metal particles comprise palladium wherein the amountof said metal particles is from about 0.001 to about 8 μg/cm².

The present invention provides use of a substrate according to thepresent invention for modifying the protein adsorption to an objectcomprising said substrate.

The present invention provides use of a substrate according to thepresent invention for modifying the bacterial adhesion to an objectcomprising said substrate.

The present invention provides use of a substrate according to thepresent invention for modifying the tissue ingrowth on an objectcomprising said substrate.

The present invention provides use of a substrate according to thepresent invention for modifying the complement activation caused by anobject comprising said substrate.

The present invention provides use of a substrate according to thepresent invention for modifying the inflammatory response caused by anobject comprising said substrate.

The present invention provides use of a substrate according to thepresent invention for modifying the blood clotting caused by an objectcomprising said substrate.

The present invention provides use of a substrate according to thepresent invention for preventing bacterial growth.

The present invention provides use of a substrate according to thepresent invention for preventing transmission of bacteria. Transmissionof bacterial infections is prevented by the prevention of thetransmission of bacteria. Examples of objects used in this context arehandles, buttons, switches, hospital equipment, surgical instruments,medical instruments, kitchen equipment, and all other objects, which areable to transmit bacteria.

The present invention provides use of a substrate according to thepresent invention for preventing transmission of a nosocomial infection.An object comprising a substrate according to the present invention canbe used in any context where it is desired to prevent transmission of abacterial infection. Preventing transmission of bacteria and thusbacterial infections will in particular prevent nosocomial infections.

Another advantage of the substrate according to the appended claims isthat it provides a possibility to modify the friction coefficient. Thusthere is provided the use of a substrate according to the presentinvention for the modification of the friction coefficient of an objectcomprising said substrate.

Other features of the invention and their associated advantages will beevident to a person skilled in the art upon reading the description andthe examples.

It is to be understood that this invention is not limited to theparticular embodiments shown here. The following examples are providedfor illustrative purposes and are not intended to limit the scope of theinvention since the scope of the present invention is limited only bythe appended claims and equivalents thereof.

EXAMPLES Example 1 Hydrophobicity of the Surface as a Function of theAmount of Metal Particles

A uniform layer of silver was deposited on a glass substrate accordingto the following method. The substrate was immersed in a cleaningsolution of chromic acid for 5 minutes at 58° C., followed by rinsing indemineralised water. The surface of the substrate was activated byimmersion in a solution of aqueous stannous chloride and then rinsed indemineralised water. The surface of the substrate was then plated with auniform layer of silver by immersion in 3 deposition solutionscomprising silver ions. This yielded a silver surface with an appliedamount of 1.2 μg/cm² corresponding to a thickness of about 115 Å.Particles consisting of 23% palladium and 77% gold were subsequentlydeposited on the first silver surface by immersion in a dilutesuspension comprising metal particles of gold/palladium. The suspensionof metal particles was made by reducing a gold salt and a palladium saltwith a reducing agent and stabilising the suspension with a stabilisingagent. The substrate was subsequently rinsed in demineralised water anddried.

Substrates with different amounts of deposited particles were made usingthe method outlined above. Amounts of particles were 0, 0.02, 0.11,0.15, and 0.19 μg/cm² respectively. For the sample with 0 μg/cm² noparticles were deposited on the surface and hence it consists of asilver surface.

The static contact angle of a drop of water in equilibrium on thedifferent substrates was measured. The advancing and receding contactangles were measured using the Wilhelmy technique.

The difference between the advancing and receding contact angle valuesis called the contact angle hysteresis and was calculated for themeasurements. The result of the experiment is depicted in Table 1.

TABLE 1 Amount of Static contact Contact angle particles anglehysteresis (μg/cm²) (degrees) (degrees) 0 52 70 0.02 50 77 0.11 56 750.15 62 80 0.19 62 84

The surface hydrophobicity of the substrate is thus modified while thesurface displays several other useful properties, such as antimicrobialproperties, inherent of the substrates according to this example.

Example 2 Protein Adsorption as a Function of the Amount of MetalParticles

A uniform layer of silver was deposited on a silicon dioxide substrate.The substrate was immersed in a cleaning solution of 20% sulphuric acidfor 10 minutes at room temperature, followed by rinsing in demineralisedwater. The surface of the substrate was activated by immersion in anaqueous solution of stannous chloride and the rinsed in demineralisedwater. The surface of the substrate was then plated with a uniform layerof silver by immersion in 4 baths of deposition solutions comprisingsilver ions. This yielded a silver surface with an applied amount of 0.8μg/cm² corresponding to a thickness of about 77 Å. Particles consistingof 95% palladium and 5% gold were subsequently deposited on the firstsilver surface by immersion in a dilute suspension of Pd/Au-particles.The applied amount of metal particles was 0.05, 0.12, 0.48 and 0.59μg/cm² respectively. The substrate was rinsed in demineralised water anddried.

Adsorption of fibrinogen was studied by the QCM-D technique. Fibrinogenis a glycoprotein synthesised in the liver and is found in blood plasma.QCM-D is a quartz crystal microbalance with dissipation monitoring.

The adsorbed amount of fibrinogen as a function of applied metalparticles is shown in table 2.

TABLE 2 Amount of Pd/Au-particles Fibrinogen adsorption (μg/cm²)(μg/cm²) 0.05 2.5 0.12 2.8 0.48 1.8 0.59 2.3

Example 3 Growth of Bacteria as a Function of the Amount of MetalParticles

Palladium/gold nanoparticles were deposited in different amounts on asilver base layer, following the method outlined in example 1. Theparticles comprised 95% palladium and 5% gold. The amount of silver inthe base layer was kept constant for all samples. Hence the amount ofdeposited Pd/Au particles was varied. The growth of bacteria as afunction of amount of deposited nanoparticles (Pd/Au) was studied usingthe following method:

Coated samples were placed into universals. Triplicates were includedfor each test condition 10 ml of artificial urine (AU) containinginoculated E. coli (roughly 10⁵ CFU/ml) was added to each one and theywere incubated horizontally with gentle shaking at 37° C. for 4 hours.After 4 hours the universals were removed from incubation. The sampleswere removed and CFU (colony forming unit) counts were done from eachuniversal by carrying out 10-fold dilutions in sterile distilled waterand plating 100 μl onto a third of a nutrient agar plate. These wereincubated for 16-24 hours at 37° C. and the colonies counted. The logCFU/ml versus a control was calculated and is shown in Table 3.

TABLE 3 Amount of nanoparticles (Pd/Au) Log CFU/ml read vs. (μg/cm²)control 0.78 6.5 0.84 7.0 1.03 6.0 1.10 6.5 1.74 5.3 2.35 4.9 2.41 4.6

Example 4 Microbial Growth for Several Species

Palladium/gold nanoparticles were deposited in different amounts on asilver base layer on a substrate of silicone, following the methodoutlined in example 1. The particles comprised 95% palladium and 5%gold. The amount of silver in the base layer was kept constant for allsamples. The amount of deposited Pd/Au particles was 0.36 μg/cm². Theantimicrobial properties for different bacterial strains were studied.

Species of microorganisms were chosen with the goal to survey a range ofcommon pathogens (clinical isolates) involved in bacteria transmissionand nosocomial infections, namely Escherichia coli (E. coli),Pseudomonas aeruginosa, Enterococcus spp, Klebsiella, and Candida.

The Pd/Au coated silicone samples were placed into universals.Triplicates were included for each test condition. 10 ml of artificialurine containing inoculated organisms (roughly 10⁵ CFU/ml) was added toeach one and they were incubated horizontally with gentle shaking at 37°C. for 24 hours.

After 24 hours the universals were removed from incubation. The sampleswere removed, drained on paper towels and then placed into universalscontaining 20 ml PBS+Tween and sonicated for 1.5 minutes.

CFU counts were done from each universal by carrying out 10-folddilutions in sterile distilled water and plating 100 μl onto a third ofa nutrient agar plate. These were incubated for 16-24 hours at 37° C.and the colonies counted. In table 4 the reduction of bacteria comparedto the uncoated silicone sample is shown. The larger the value thegreater reduction.

TABLE 4 Reduction vs. Control (Log CFU/cm) E. coli PseudomonasEnterococcus Klebsiella Candida Uncoated Silicone 0.00 0.00 0.00 0.000.00 Pd/Au particle coated 1.64 2.53 3.88 1.37 2.52 silicone

Example 5

A net of polyester fabric was first rinsed in a 5% potassium hydroxidesolution for 5 min at 30° C. After repeated rinsing in demineralisedwater the substrate was immersed in an acidified solution of 1 g/lstannous chloride at room temperature for 10 min. After rinsing indemineralised water it was soaked in a plating bath containing 2 g/lcopper sulphate, 5 g/l sodium hydroxide, 50 g/l sodium citrate and 0.005ml/l formaldehyde for 10 min at 35° C. A copper layer of about 200 Å wasobtained and after new rinsing in demineralised water the substrate wasimmersed in a particle suspension comprising 0.05 g/l each of palladiumparticles and gold particles. The applied amount of metal particles was0.4 μg/cm².

Example 6

A substrate of PMMA was cleaned in 5% hydrochloric acid for 2 min andthen rinsed in demineralised water before dipping in a solutioncontaining 0.02 g/l of the stannous ion at a pH of 2.5. After rinsingthe substrate was immersed in a solution containing 0.005 g/l of silverions, 0.02 ml/l ammonia, 0.05 g/l potassium hydroxide and 0.0005 ml/lformaldehyde for 5 min at room temperature. This gave a surface with0.12 μg/cm² of silver. After rinsing it was immersed in a particlesuspension comprising 0.005 g/l palladium and 0.002 g/l gold particles.The applied amount of metal particles was 0.05 μg/cm².

Example 7

A non-woven polyimide substrate was immersed in a 12% solution of NaOHat 40° C. for 10 min. After repeated rinsing in demineralised water itwas immersed in an alcoholic solution containing 0.5 g/l stannouschloride for 5 min at room temperature. After rinsing it was soaked in acopper bath according to example 3. A copper layer of 2 μg/cm² wasobtained. After rinsing it was immersed in a suspension comprising 1% ofPd and 0.2% of gold particles, calculated on the weight of the totalsuspension. The applied amount of metal particles was 0.6 μg/cm².

Example 8

A nylon fabric was cleaned in 5% NaOH for 10 min at 40° C. and afterrinsing in demineralised water immersed in a solution of 0.6 g/lstannous chloride at pH 2.2 for 15 min at room temperature. After thisthe surface comprised a silver amount of 0.8 μg/cm². After a new rinsingit was dipped in a silver bath according to example 2 and then after newrinsing dipped in a suspension comprising 1% Pd and 0.05% Au particles.The applied amount of metal particles was 0.12 μg/cm².

Example 9

A substrate of aluminium was treated in a solution of 10% nitric acidand 3% hydrofluoric acid at 60° C. for 20 min. After rinsing, thesubstrate was dipped in an acidified solution of 3 g/l stannous chlorideand after renewed rinsing in a silver bath according to example 2. Afterthis step an amount of around 80 Å silver was obtained on the surface.After another rinsing the substrate was immersed in a suspensioncomprising 1% Pd and 2% Au particles. The applied amount of metalparticles was 0.7 μg/cm².

Example 10

A substrate of PTFE was etched in an aqueous solution of sodiumhydroxide for 5 min. After rinsing and drying it was immersed in asolution containing 0.7 g/l stannous chloride for 20 min at roomtemperature. The substrate was after rinsing dipped in a plating bathcontaining 0.2 g/l silver nitrate, 0.5 ml/l ammonia and sodium hydroxideto pH 10.5 for 5 min. After this step an amount of around 2.2 μg/cmsilver was obtained on the surface. After a new rinse it was immersed ina suspension comprising 3% Pd and 0.1% Au particles for 5 min at roomtemperature. The applied amount of metal particles was 0.03 μg/cm².

Example 11

A glass plate was rinsed in 10% sulphuric acid and 1% hydrofluoric acidat room temperature for 15 min. After rinsing it was immersed in a 1%stannous fluoride solution and after a new rinse immersed in a silverbath according to example 2. After this step an amount of around 140 Åsilver was obtained on the surface. After renewed rinsing it was dippedin a suspension comprising 1% ruthenium and 2% palladium particles. Theapplied amount of metal particles was 0.25 μg/cm².

Example 12

A stainless steel substrate was immersed in a solution of 15% nitricacid and 5% HF at room temperature for 30 min and then rinsed indemineralised water. The process continued following the steps inexample 11. The applied amount of metal particles was 0.9 μg/cm².

Example 13

A titanium rod was cleaned in a solution of 18% nitric acid and 2% HFfor 20 min at room temperature. The application of an electron donatingsurface and the application of metal particles was made as in example11. The applied amount of metal particles was 0.6 μg/cm².

Example 14 Complement Activation

Detection of Surface Induced Complement Activation with Quartz CrystalMicrobalance with Dissipation Monitoring (QCM-D)

The quantification of a foreign body response is indirectly achieved bymonitoring the binding of rabbit-anti human antibodies directed to thesurface bound complement factor C3b.

Within seconds from introduction to a soft tissue a foreign body issubject to great attention from the complement system. The complementsystem comprises about 30 different proteins where C3 is the mostabundant. After high concentration body fluid proteins (i.e. Albumin,Fibrinogen and Fibronectin) the complement system is one of the firstactors on the scene and aims to protect the host from invading bacteriaand fungi, but also to alert the immune system about a foreign bodyentering the system.

Without being bound by any specific scientific theory the inventorsassume that when complement factor 3 (C3) binds to an introduced surfaceit is cleaved by C3 convertase to form soluble C3a, and surface boundC3b. The surface bound C3b will then act as a convertase itself,triggering subsequent cleavage of C3 in a cascade-like fashion.Receptors to C3b is found on erythrocytes, macrophages, monocytes,polymorphonulear leukocytes and B cells, all of which are important incontrolling inflammation and wound healing in tissue. The exactmechanisms controlling the binding of C3 to the surface are still muchunknown. However, antibodies directed specifically towards C3b caneasily be measured in vitro with QCM-D and give quantitative informationof a biomaterial's immune response properties. This new methodology showgood agreement with all other known methods for the detection of surfacebound C3b.

Material and Methods Preparation of Surfaces

A coating was applied on standard SiO₂ QCM-D crystals (QSX 303, Q-SenseSweden) using the method outlined in example 2.

As model surfaces standard QCM-D crystals sputtered with Au (s), Ti (s)(QSX301 and QSX310 respectively) was used.

Ag (s) and Pd (s) model surfaces were made on standard gold platedQCM-D-D crystals (QSX 301, Q-Sense Sweden) by high vacuum sputtering ofapproximately 200 Å Palladium and Silver respectively.

Blood Products

We received fresh whole blood from five healthy donors (SahlgrenskaUniversity Hospital, Göteborg, Sweden). The blood was let to clot inroom temperature for approximately 4 hours to obtain complement activeserum. The serum was then centrifuged at 4000 rpm for 20 min (HettichUniversal 16 R) after which the supernatant was removed andre-centrifuged as above and stored at −70° C.

Detection of surface induced complement activation Serum was diluted 1:5in Veronal Buffer Saline supplemented with CaCl₂ (0, 15 mM) and MgCl₂(0, 5 mM) (VBS⁺⁺), and the adsorption of serum proteins to the modifiedQCM-D-crystals were monitored for 20 minutes followed by a rinse withbuffer for 5 minutes. The rinse was followed by the addition ofrabbit-anti-human C3b antibodies diluted 1:20 in VBS⁺⁺ (Sigma). Fornegative and positive controls, standard gold QCM-D crystals pre-coatedwith human IgG (1 mg/ml) (Sigma) were used. The negative control washeat inactivated at 56° C. for 30 min prior to measurements.

All experiments were carried out at room temperature in Veronal BufferSaline with CaCl₂ (0, 15 mM) and MgCl₂ (0, 5 mM) (VBS⁺⁺) except fornegative controls were VBS⁻⁻ were used. All QCM-D measurements werepreformed on the apparatus D300 (Q-sense, Sweden).

Results of the QCM-D measurements of complement activation (C3b). TheSiO₂ surfaces coated as described above had an amound of silver of0.35-0.61 μg/cm². The amount of gold in the particles was variedaccording to the table below and the complement activation was measuredaccording to the table.

Amount of C3b Sample Au (μg/cm²) (ng/cm²) Sample No 1 0.09 677 Sample No2 0.41 991 Neg control — 109 Pos control — 1832 Titan — 632 (control)

Example 15 Platelet Adhesion and Soluble Complement Factor C3aProduction on Biomaterial Surfaces

The consumption of platelets in fresh whole blood exposed to abiomaterial is used to quantify the thrombogenicity of a desiredbiomaterial. Moreover, the soluble fraction of activated complementfactor 3 (C3a) is used to monitor the complement activation from thebiomaterial surface.

Background

Platelets (or thrombocytes) are small disc-shaped anuclear cellfragments normally present in healthy blood. They play a crucial role inpreserving the walls in blood vessels and are recruited to a damagedarea and activated to form a plug, preventing hemorrhage and blood loss.Platelets are also known to adhere and become activated on certainbiomaterial surfaces, sometimes forming an undesired and potentiallyhazardous clot.

Soluble C3a is a small protein cleaved off from the complement factor 3(C3) when this is bound and activated on a bacteria or a foreign bodysurface. C3a acts as a chemo-attractant for polymorphonuclear monocytesand also have anaphylatoxic properties signaling for the release ofhistamine from mast cells.

Material and Methods Experimental Chambers

The experimental chamber is briefly constructed of two PMMA rings gluedonto a PMMA microscopic slide, constructing two wells. After addition ofwhole blood, the material to be tested is placed as a lid over the twowells and held in position with a clip. The chamber is then mounted on adisc rotating in 37° C. water for 60 minutes at 22 rpm.

Blood

Blood was drawn from one healthy donor and collected in a 2× heparinizedvial containing soluble heparin (Leo Pharma), to give a finalconcentration of 1.0 IU heparin/ml. The collected blood was thenimmediately transferred to the experimental chambers.

Platelet Counting

After incubation in the experimental chamber the blood was added EDTA(Fluka) to a final concentration of 4 mM. Platelets were then counted ona Coulter AcT Diff™ (Coulter Corporation) automated cell counter.

C3a Analysis

After platelet counting, the blood was centrifuged at 4600 g for 10 minat +4° C. and the supernatant (plasma) was saved and stored in −70° C.prior to measurements.

Plasma was diluted 1/300 and analysed in a sandwich ELISA which employsthe monoclonal 4SD17.3 (Uppsala university, Sweden) as capture antibody.Bound C3a was detected with biotinylated rabbit anti-human C3a (Dako),followed by HRP-conjugated streptavidin (Amersham Biosciences).Zymosan-activated serum, calibrated against a solution of purified C3a,served as a standard.

Results

Blood platelet count and C3a adsorption. The coated objects manufacturedfollowing the method outlined I example 2 on glass had a silver surfaceconcentration of about 1.3 μg/cm2

Amount of Number of palladium platelets C3a Sample (μg/cm²) (×10⁹)(μg/ml) Uncoated Glass — 29 681 coating 0 170 337 variation 1 coating0.01 190 287 variation 2

Blood platelet count and C3a adsorption. The coatings on glass had asilver surface concentration of about 1.3 μg/cm².

Number of Amount of gold platelets C3a Sample (μg/cm²) (×10⁹) (μg/ml)Uncoated Glass — 29 681 Coating 0.01 166 376 variation 3 coating 0.01141 271 variation 4

Example 16 Measurement of Inflammatory Response Material

NHSp-2 (Normal human serum pool from Immunologisk institutt,Rikshospitalet, Oslo, Norway), serum from healthy blood donors.

30 cm PDMS (Polydimethylsiloxane) tubes were coated according to theprocedure outlined in example 1. PVC tubes 30 cm were used as control.

Setup: 7 types of tubes, untreated and PVC in triplicate (in total 21).

Method:

-   -   1) The serum was placed on ice.    -   2) A zero sample was removed. 750 μl was added directly to a        tube with 15 μl EDTA 0.5M. The sample was kept on ice.    -   3) 750 μl serum was added to each tube.    -   4) The tubes were attached to a rotor (5 rpm) 37° C. and were        incubated for 30 minutes.    -   5) The serum was removed with a pipette and added to a tube with        15 μl EDTA 0.5M. The samples were placed on ice and analysed        with respect to TCC (the soluble terminal C5b-9 complement        complex).

TCC was analyzed using a double antibody enzyme immunoassay based on themonoclonal aE11 antibody, highly specific for a neoepitope exposed onactivated but not native C9, as catching antibody. The method wasoriginally described in:

-   Mollnes T E, Lea T, Frøland S S, Harboe M. “Quantification of the    terminal complement complex in human plasma by an enzyme-linked    immunosorbent assay based on monoclonal antibodies against a    neoantigen of the complex”, Scand J Immunol 22:197-202. 1985.    and later modified in:-   Mollnes T E, Redl H, Høg>sen K, Bengtsson A, Garred P, Speilberg L,    Lea T, Oppermann M, Götze O, Schlag G. “Complement activation in    septic baboons detected by neoepitope specific assays for    C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC)”,    Clin Exp Immunol 91:295-300. 1993.

Results

Inflammatory response, amount of Ag=1 μg/cm², coated according to themethod outlined in example 2 on a PDMS tube.

Sample Amount Pd TCC IL-8 No (μg/cm²) (pg/ml) (pg/ml) 1 0.47 5.851582.13 2 0.70 5.16 1724.33 3 1.48 4.60 2136.79 Uncoated Uncoated 3.88728.33 PDMS tube Uncoated Uncoated 6.21 1750.23 PVC tube, control

Inflammatory response, amount of Ag=1 μg/cm², coated according to themethod outlined in example 2 on a PDMS tube.

Sample Amount Au IL-8 No (μg/cm²) TCC (pg/ml) (pg/ml) 4 0.14 5.241652.62 5 0.32 6.51 1264.40 Uncoated Uncoated 3.88 728.33 PDMS tubeUncoated Uncoated 6.21 1750.23 PVC tube, control

Example 17 In Vitro Method

Primary normal human dermal fibroblasts (NHDF, Karocell TissueEngineering AB, Stockholm, Sweden), passage 7, were used. The cells werecultured in tissue culture flasks in complete fibroblast mediumcontaining DMEM+GlutaMAX™−1 (Gibco, UK), 10% foetal bovine serum (FBS,Gibco, UK) and 1% Antibiotic-Antimyocotic (Gibco, UK) at 37° C., 5% CO₂and 95% humidity. Ten different coated materials (PDMS) preparedaccording to the method outlined in example 1 were sterilely punchedinto discs with a diameter of 15 mm to fit in a 24-well plate. Discswere dipped in sterile PBS (Phospate buffered saline solution, Gibco,UK) and 1 ml of cell suspension (17000 cells/ml) was dispersed over thedisks and in empty PS-wells (polystyrene, Falcon, BD Biosciences,Belgium) and incubated for 24 h and 72 h in triplicates. Medium from allsamples were collected, centrifuged at 400 g, 5 min and stored at −70°C. for ELISA (enzyme-linked immunosorbent assay) analyses of cellreleased factors. Two discs of each material were incubated withcomplete medium without cells to estimate background values.

Cell Amount

Cell amounts in association with the surfaces and surrounding mediumwere determined by a NucleoCounter®-system (ChemoMetec A/S, Denmark).Briefly, cells were treated with lysis buffer and stabilizing buffer(provided with the system). Lysed samples were loaded in aNucleoCassette® precoated with fluorescent propidium iodide that stainsthe cell nuclei, and were then quantified in the NucleoCounter®.

Cell Viability

Cell viability was determined by measuring lactate dehydrogenase content(LDH) in medium, a marker of cell membrane injury, using aspectrophotometric evaluation of LDH mediated conversion of pyruvic acidto lactic acid (C-Laboratory, Sahlgrenska University Hospital, Göteborg,Sweden).

Cytokine Determination

The amount of TGF-β1 (Transforming Growth Factor beta 1) and type Icollagen were detected by ELISA kits (Human TGF-β1, Quantikine®, R&DSystems, UK; Human collagen type1 ELISA KIT, Cosmo Bio Co., Japan)according to the manufacturer's instruction, in a SpectraVmax ELISAreader (Molecular Devices, UK).

In Vivo Method

Six different coated PDMS objects (10 mm in diameter) were coatedaccording to the method outlined in example 1 and were sterilized.Female Spraque-Dawley rats (200-250 g), fed on a standard pellet dietand water were anaesthetized with a mixture of 2.7% isofluran and air(Univentor 400 Anaesthesia Unit, Univentor, Malta) and 0.01 mg Temgesicwas given as analgesic s.c. pre-operatively. Rats were shaved andcleaned with 5 mg/ml chlorohexidine in 70% ethanol and each rat receivedone of each implant type subcutaneously (s.c.) on the back. The woundswere closed with 2 sutures (Ethilon 5-0 FS-3, Ethicon®, Johnson &Johnson, Belgium). The implantation periods were 1 and 3 days toevaluate the early inflammatory process and 21 days for the examinationof the fibrous capsule formation and the late inflammatory response (n=8rats per time period). When the explantation was performed the animalswere sacrificed by an overdose of pentobarbital (60 gL⁻¹) after shortanaesthetics with a mixture of 2.7% isofluran and air. The implants andthe surrounding exudates were retrieved. The exudate cells were obtainedfrom the pockets by repeated aspiration of HBSS (Hank's balanced saltsolution, Gibco, UK) and kept on ice. The exudates were centrifuged at400 g, 5 min and supernatants were kept at −70° C. All implantationstudies were approved by the Local Ethical Committee for LaboratoryAnimals.

Cell Amount and Cell Type

The concentration and type of cells in the exudates (cells/ml) werecounted by light microscopy with Türk staining in a Bürker chamber andcell amount in centrifuged exudates and on implants were determined byNucleoCounter®-system.

Cell Viability

Cell viability was determined by Trypan Blue exclusion using lightmicroscopy and by LDH evaluation (C-Laboratory, Sahlgrenska UniversityHospital, Göteborg, Sweden).

Cytokine Determination

The amount of TGF-β1 (Transforming Growth Factor beta 1) and MCP-1(Monocyte Chemoattractant Protein-1) were detected by ELISA kits (RatTGF-β1, Quantikine®, R&D Systems, UK; Amersham Monocyte ChemoattractantProtein-1 [(r)MCP-1], Rat, Biotrak ELISA System, GE Healthcare, UK)according to the manufacturer's instruction, in a SpectraVmax ELISAreader (Molecular Devices, UK).

Results from the In Vitro Study

The amount of metals on the test object of PDMS coated according to themethod outlined in example 2 was Ag=0.8-0.9 μg/cm² and Pd=0.1 μg/cm².

Surface concentration of Au Number of cells after (μg/cm²) 72 h 0.055500 0.34 9400 0.43 16200

In the second experimental set the amount of metals on the test objectof PDMS coated according to the method outlined in example 2 wasAg=0.8-0.9 μg/cm² and Au=0.05-0.09 μg/cm².

Surface concentration of Pd Number of cells after (μg/cm²) 72 h 0.1 55000.27 8600 0.69 9900Results from the In Vivo Study

The Amount of Pd was verified on PDMS discs in vivo. The amount of Agwas about 1 μg/cm² for all samples. (PMN=polymorphonuclear)

Amount of % PMN cells % PMN cells % PMN cells Pd in exudate, in exudate,in exudate, (μg/cm²) 1 day 3 days 21 days Uncoated 33 2 1 PDMS control 017 2 1 0.07 32 2, 5 Below 1 0.86 26 2 Below 1

Total Total Total amount of amount of Amount of amount of MCP-1, 3MCP-1, 21 Pd MCP-1, 1 days days (μg/cm²) day (pg/ml) (pg/ml) (pg/ml)Uncoated 4600 500 100 PDMS control 0 2050 700 350 0.07 4500 500 200 0.863300 600 200

Total Total Total amount of amount of Amount of amount of TGF-1, 3TGF-1, 21 Pd TGF-1, 1 days days (μg/cm²) day (pg/ml) (pg/ml) (pg/ml)Uncoated 62 1 10 PDMS control 0 3 8 12 0.07 82 33 11 0.86 25 8 20

The Amount of Au was varied on discs in vivo. The amount of Ag was about1 μg/cm² for all samples. (PMN=polymorphonuclear)

Amount % PMN cells % PMN cells % PMN cells of Au in exudate, in exudate,in exudate, (μg/cm²) 1 day 3 days 21 days Uncoated 33 2 1 PDMS control0.01 32 2.5 1 0.43 18 5 1.5 0.64 20 4 Below 1

Total Total Total amount of amount of amount of MCP-1, 3 MCP-1, 21Amount of MCP-1, 1 days days Au (μg/cm²) day (pg/ml) (pg/ml) (pg/ml)Uncoated 4600 500 100 PDMS control 0.01 4500 500 200 0.43 3100 450 2000.64 2800 500 150

Total Total Total amount of amount of amount of TGF-1, 3 TGF-1, 21Amount of TGF-1, 1 days days Au (μg/cm²) day (pg/ml) (pg/ml) (pg/ml)Uncoated 62 1 10 PDMS control 0.01 82 33 11 0.43 6 5 20 0.64 28 8 9

Below are described a number of specific uses of the coating accordingto the present invention.

Contact Lenses

Contact lenses are often made of a polymeric material with significantwater content. It is essential to avoid microbial growth on a contactlens. By using the method outlined above it is possible to coat acontact lens to prevent or reduce microbial growth. A coated contactlens will also be biocompatible. In the examples above it has beendemonstrated that polymeric material can be coated according to theinvention. As examples of coating of polymeric substrates can bementioned coating of polyester (example 5), PMMA (example 6), polyimide(example 7), nylon (example 8), and PTFE (example 9). The fact that thecoating successfully can be applied to those polymeric materials showsthat the coating also can be applied to contact lenses of polymericmaterials.

Pacemakers, and Pacemaker Electrodes

Pacemakers to be inserted into the body of a human have to bebiocompatible. At the same time it is desirable if they preventmicrobial growth. A pacemaker or pacemaker electrode coated with thepresent coating has those desirable properties. Above it has been shownthat the coating can be applied to many materials, for instance metalssuch as titanium (example 13), stainless steel (example 12), andaluminium (example 9). Thus a pacemaker or pacemaker electrode made ofmetal or any other material can successfully be coated according to thepresent invention.

Stents (Bare Metal and Drug Eluting)

Stents to be inserted into the body of a human should preferably bebiocompatible. At the same time it is desirable if they preventmicrobial growth. A stent coated with the present coating has thosedesirable properties. Above it has been shown that the coating can beapplied to metals such as titanium (example 13), stainless steel(example 12), and aluminium (example 9). Stents may be manufactured ofthese and other metals or alloys and may successfully be coated with thecoating according to the present inventions.

Dental Implants

Dental implants are advantageously both biocompatible and antimicrobial.Dental implants can be made of titanium or any other materials. As shownabove in example 13, titanium can be coated according to the presentinvention. A dental implant coated according to the present invention isboth biocompatible and antimicrobial. One example of a dental implant isa dental implant made of titanium and coated as described in example 13.

Rupture Nets, Mesh

Materials for nets and meshes can be coated as shown for polyester(example 5), PMMA, (example 6), polyimide (example 7), and nylon(example 8). Such nets and meshes will be both antimicrobial andbiocompatible which is an advantages within many applications.

Blood Centrifuge Equipment (in Contact with Blood)

In equipment intended for contact with blood the biocompatible andantimicrobial properties of the coating according to the presentinvention are desired. Materials in contact with blood can be selectedfrom a large number of materials. In the examples above we have shownthat a large variety of materials can be coated, such as glass (example1, 2, 4, and 11), polyester (example 5), PMMA, (example 6), polyimide(example 7), nylon (example 8), aluminium (example 9), PTFE (example10), stainless steel (example 10), and titanium (example 13). Bloodcentrifuge equipment comprising a substrate coated according to thepresent invention has improved properties regarding biocompatibility andantimicrobial properties.

Surgical Instruments

It is highly desirable that surgical instruments display antimicrobialproperties. Materials often used for surgical instruments such asstainless steel and titanium can be coated as shown in examples 12 and13 respectively. By using the coating according to the present inventionthe desired antimicrobial properties are achieved. Moreover the coatingis also biocompatible.

Gloves

It is often desired that gloves used for various purposes displayantimicrobial properties. Moreover gloves which at the same time aretissue friendly and biocompatible are desired for some applications. Bycoating gloves with the coating according to the present invention theabove mentioned desired properties are achieved. Polymeric materials canbe coated according to the present invention with excellent results andexamples of several polymeric materials are given above.

Blood Bags

In blood bags intended for contact with blood the biocompatible andantimicrobial properties of the coating according to the presentinvention are desired. Materials for blood bags are most often polymericmaterials. Polymeric materials can be coated according to the presentinvention with excellent results and examples of several polymericmaterials are given above.

Artificial Heart Valves

For artificial heart valves the antimicrobial and biocompatibleproperties of the coating according to the present invention are highlydesired. The coating can be applied successfully both to polymericmaterials and metals that may constitute an artificial heart valve. Theabove mentioned examples show that the coating can be applied to bothpolymeric materials and metals as well as alloys.

Central Venous Catheters

For catheters to be inserted into the body such as central venouscatheters, antimicrobial properties are highly desired. Moreover objectsto be inserted into the human body also should be biocompatible andtissue friendly. The coating according the present invention fulfils therequirements and has excellent properties for catheters. Materials usedfor catheters can be coated successfully with the coating according tothe present invention.

Peripheral Venous Catheters

Regarding antimicrobial and biocompatible properties the requirementsfor peripheral venous catheters and central venous catheters aresimilar. Thus the coating according to the present invention is alsoexcellent for peripheral venous catheters.

Vascular Ports

Regarding vascular ports there is an infection risk and moreover suchvascular ports should be biocompatible. Therefore the coating accordingto the present invention is excellent for vascular ports so that theybecome antimicrobial and biocompatible. Materials used for vascularports can successfully be coated with the coating according to thepresent invention.

Haemodialysis Equipment

For haemodialysis equipment antimicrobial and biocompatible propertiesare important, thus making the coating according to the presentinvention very suitable.

Peritoneal Dialysis Equipment

For peritoneal dialysis equipment the antimicrobial and biocompatibleproperties of the coating according to the present invention are veryuseful. It is suitable to apply the coating according to the presentinvention to parts of such equipment.

Plasmapheresis Devices

For plasmapheresis devices, including catheters implanted for suchpurpose, the coating according to the present invention is suitable dueto its antimicrobial and biocompatible properties. Materials used inthis context can successfully be coated according to the presentinvention.

Inhalation Drug Delivery Devices

Inhalation drug delivery devices advantageously display antimicrobialproperties which is achieved by coating suitable parts of the devicewith the coating according to the present invention. The biocompatibleproperties of the coating is also an advantage.

Vascular Grafts (for Example Arterial Grafts)

Vascular grafts benefit from antimicrobial and biocompatible properties,which are achieved by the coating according to the present invention.The materials which the grafts are made of are suitable for coatingaccording to the present invention.

Cardiac Assist Devices

Cardiac assist devices to be implanted into the body should be bothbiocompatible and antimicrobial. This is achieved by using a coatingaccording to the present invention. Materials used for such devices aresuccessfully coated using the present invention.

Wound Dressings

Wound dressings are preferably antimicrobial as well as biocompatible.This make them excellent objects for coating according to the presentinvention. Polymeric and fibrous material used for wound dressings aresuccessfully coated according to the present invention.

Intermittent Catheters

Intermittent catheters as well as other catheters should preferably beboth antimicrobial to avoid problems with infections, moreover theyshould also be biocompatible. The coating according to the presentinvention is excellent for catheters since it is both antimicrobial andbiocompatible. Materials used for catheters can successfully be coatedaccording to the present invention.

ECG Electrodes

ECG electrodes should preferably be both antimicrobial andbiocompatible. ECG electrodes coated according to the present inventionare both antimicrobial and biocompatible. Materials such as titanium(example 13), stainless steel (example 12), and aluminium (example 9)can as well as many other materials suitable for electrodes be coatedaccording to the present invention.

Peripheral Stents

Desired properties for peripheral stents are similar to those for stentsas described above. Thus also peripheral stents can successfully becoated according to the present invention.

Bone Replacing Implants

Implants of different kinds such as bone replacing implants arepreferably both antimicrobial and biocompatible. This is achieved by acoating according to the present invention.

Orthopaedic Implants

Orthopaedic implants as are very suitable to coat according to thepresent invention to render them antimicrobial and biocompatible.Examples of orthopaedic implants include hip replacements, total hipreplacements, ceramic hip replacements, hip joint replacements, kneereplacements, total knee replacements, and knee joint replacements.

Orthopaedic Devices (Screws, Pins, Staples, Suture Anchors Etc)

All kinds of orthopaedic devices such as screws, pins, staples, andsuture anchors are preferably both antimicrobial and biocompatible. Suchdevices are made of materials which successfully can be coated accordingto the present invention. Orthopaedic devices benefit from coatingaccording to the present invention. One example of an orthopaedic devicea screw of titanium coated according to the procedure described inexample 13.

Tissue Replacing Implants

Implants of different kinds such as tissue replacing implants areadvantageously both antimicrobial and biocompatible. This is achieved bya coating according to the present invention on the tissue replacingimplants.

Intraocular Lenses

For intraocular lenses it is an advantage if they are antimicrobial andbiocompatible. This is achieved by coating according to the presentinvention. Intraocular lenses made of polymeric materials and othermaterials can successfully be coated according to the present invention.

Sutures

It is a great advantage for sutures to be antimicrobial andbiocompatible. Sutures are therefore suitable for coating according tothe present invention.

Needles

Needles that should be antimicrobial and/or biocompatible cansuccessfully be coated according to the present invention to give thedesired antimicrobial and biocompatible properties.

Drug Delivery Devices

Drug delivery devices which shall be made antimicrobial and/orbiocompatible are advantageously coated according to the presentinvention.

Endotracheal Tubes

Endotracheal tubes are preferably antimicrobial as well asbiocompatible. The polymeric materials that are used to manufactureendotracheal tubes are suitable for coating according to the presentinvention. Thus endotracheal tubes can successfully be coated accordingto the present invention to give the desired antimicrobial andbiocompatible properties.

Shunts

For various kinds of shunts it is highly desirable that they displayantimicrobial properties and that they are biocompatible. The materialsthat are used for shunts can successfully be coated according to thepresent invention and thus the shunt will get the desired properties.

Drains

Drains are preferably antimicrobial and also biocompatible. Since thecoating according to the present invention successfully can be appliedto the materials from which drains are made, it is very suitable toapply the coating according to the present invention to drains.

Suction Devices

Suction devices should be antimicrobial and also biocompatible. Sincethe coating according to the present invention successfully can beapplied to the materials from which suction devices are made, it is verydesirable to apply the coating according to the present invention tosuction devices.

Hearing Aid Devices

Hearing aid devices are preferably antimicrobial and also biocompatible.The materials that hearing aid devices are made from can successfully becoated according to the present invention. Hearing aid devices are verysuitable to coat according to the present invention.

Urethral medical devices such as catheters, urethral stents andsuprapubic stents are suitable to coat according to the presentinvention.

Artificial blood vessels are suitable to coat according to the presentinvention.

1. A substrate having an electron donating surface, characterized inthat there are metal particles on said surface, said metal particlescomprise palladium and at least one metal selected from the groupconsisting of gold, ruthenium, rhodium, osmium, iridium, and platinumand wherein the amount of said metal particles is from about 0.001 toabout 8 μg/cm².
 2. The substrate according to claim 1, wherein saidelectron donating surface is a layer of an electron donating materialwhich is applied in an amount of about 0.05 to about 12 μg/cm².
 3. Thesubstrate according to claim 2, wherein said electron donating layer isa metal that is less noble than any of the metals in the groupconsisting of palladium, gold, ruthenium, rhodium, osmium, iridium, andplatinum.
 4. The substrate according to claim 3, wherein said electrondonating layer is a metal selected from the group consisting of silver,copper and zinc.
 5. The substrate according to claim 1, wherein saidsubstrate is a polymeric substrate.
 6. The substrate according to claim5, wherein said polymeric substrate is selected from the groupconsisting of latex, vinyl, polymers comprising vinyl groups,polyurethane urea, silicone, polyvinylchloride, polypropylene, styrene,polyurethane, polyester, copolymerisates of ethylene vinyl acetate,polystyrene, polycarbonate, polyethylene, polyacrylate,polymethacrylate, acrylonitrile butadiene styrene, polyamide, andpolyimide, or mixtures thereof.
 7. The substrate according to claim 5,wherein said polymeric substrate is selected from the group consistingof a natural polymer, a degradable polymer, an edible polymer, abiodegradable polymer, an environmental friendly polymer, and a medicalgrade polymer.
 8. The substrate according to claim 1, wherein saidsubstrate is a metal.
 9. The substrate according to claim 8, whereinsaid metal is selected from the group consisting of stainless steel,medical grade steel, titanium, medical grade titanium, cobalt, andchromium or mixtures thereof.
 10. The substrate according to claim 1,wherein said substrate is selected from the group consisting of glass,minerals, zeolites, stone and ceramics.
 11. The substrate according toclaim 1, wherein said substrate is selected from the group consisting ofpaper, wood, woven fibres, fibres, cellulose fibres, leather, carbon,carbon fibres, graphite, polytetrafluoroethylene, andpolyparaphenyleneterephthalamide.
 12. The substrate according to claim1, wherein said substrate has the shape of a particle.
 13. The substrateaccording to claim 1, wherein the amount of the metal particles is fromabout 0.01 to about 4 μg/cm².
 14. The substrate according to claim 1,wherein the ratio of palladium to non-palladium metals in said metalparticles is from about 0.01:99.99 to about 99.99:0.01.
 15. Thesubstrate according to claim 1, wherein the ratio of palladium tonon-palladium metals in said metal particles is from about 0.5:99.5 toabout 99.8:0.2.
 16. The substrate according to claim 1, wherein theratio of palladium to non-palladium metals in said metal particles isfrom about 2:98 to about 95:5.
 17. The substrate according to claim 1,wherein said metal particles, in addition to palladium, comprise gold.18. The substrate according to claim 1, wherein said metal particleshave an average size of about 10-10000 Å.
 19. The substrate according toclaim 1, wherein said metal particles have an average size of about100-600 Å.
 20. A medical device comprising a substrate according toclaim 1 wherein the medical device is selected from the group consistingof: contact lenses, pacemakers, pacemaker electrodes, stents, dentalimplants, rupture nets, rupture mesh, blood centrifuge equipment,surgical instruments, gloves, blood bags, artificial heart valves,central venous catheters, peripheral venous catheters, vascular ports,haemodialysis equipment, peritoneal dialysis equipment, plasmapheresisdevices, inhalation drug delivery devices, vascular grafts, arterialgrafts, cardiac assist devices, wound dressings, intermittent catheters,ECG electrodes, peripheral stents, bone replacing implants, orthopaedicimplants, orthopaedic devices, tissue replacing implants, intraocularlenses, sutures, needles, drug delivery devices, endotracheal tubes,shunts, drains, suction devices, hearing aid devices, urethral medicaldevices, and artificial blood vessels. 21-58. (canceled)
 59. A method ofuse of said substrate according to claim 1, the method comprisingintegrating said substrate into a medical device, wherein the substrateexhibits a property selected from the group consisting of: modifyingprotein adsorption to an object comprising said substrate, modifyingbacterial adhesion to an object comprising said substrate, modifyingtissue ingrowth on an object comprising said substrate, modifyingcomplement activation caused by an object comprising said substrate,modifying inflammatory response caused by an object comprising saidsubstrate, modifying blood clotting caused by an object comprising saidsubstrate, preventing bacterial growth, preventing transmission ofbacteria, preventing nosocomial infections, modifying the frictioncoefficient of an object comprising said substrate, and modifying thesurface hardness of an object comprising said substrate. 60-69.(canceled)
 70. A method for the manufacture of a substrate according toclaim 1 comprising the steps: a. depositing metal particles from asuspension onto said substrate, b. rinsing said substrate, and c. dryingsaid substrate.
 71. The method according to claim 68 further comprisingthe step of depositing an electron donating material on said substratebefore the deposition of metal particles.